In this post-genomic era, a comprehensive research of proteins becomes more and more important, and a rapid and accurate technology for identifying a protein is needed. One of methods employed by many researchers is a technology for isolating an intended protein from various proteins by two-dimensional gel electrophoresis followed by analyzing the protein by mass spectrometry. With respect to the methods of mass spectrometry, development of an electrospray ionization (ESI) method and a matrix-assisted laser desorption/ionization (MALDI) method resulted in an establishment of a soft ionization technology of biopolymers, which leads to an innovative advancement of a proteomics research.
In MALDI mass spectrometry, a high molecular weight sample such as a protein is mixed with a matrix (for example, sinapinic acid) and subjected to a co-crystallization. The matrix serves to absorb a laser light and transfer its energy to the high molecular weight sample. The high molecular weight sample is ionized and typically produces an ion species of a (M+H)+ type. Through this soft laser desorption process, the high molecular weight sample transfers into a gas phase. In the case of a time of flight (TOF) detector, upon ionization of the high molecular weight sample, the ion is accelerated in the electric field, and then arrives at the detector, where the time span from the ionization and the detection is measured and calculated at a high accuracy. The mass can accurately be calculated since the time of flight of an ion depends on the momentum, the square root of the mass-to-charge ratio (m/z).
In an ESI method, a liquid containing a high molecular weight sample eluted from a high pressure liquid chromatography (HPLC) column or a syringe pump at a microvolume/time is nebulized and injected into an electrospray ion source to effect an ionization. The ESI method is becoming a main stream of the proteomics analysis since its sample preparation is automated in advance of the MALDI method.
On the other hand, as a method for preparing a sample for the MALDI method, it is mainly employed to prepare a co-crystal with a matrix on a target plate after purifying the sample by a simple chromatography using reverse phased particles. However this method requires a technical skill and involves significant problems for attempting automation. Alternatively, there is a report that the surface of the target plate is modified with a self-assembled monolayer and impurities are removed on the target plate (for example, see non-patent references 1 and 2). Any of these methods is based on the concept that the impurities are removed before forming a co-crystal of the sample with a matrix. In addition, a method in which a chromatography medium is added for the purpose of removing salts mainly on a target plate (for example, see non-patent reference 3), or a two-layer method in which on a lower matrix formed using a reagent capable of ion-pairing with SDS an upper matrix is formed using an SDS-containing sample (ion-pair assisted recovery, for example, see non-patent reference 4), but any of these may fail to show a sufficient effectiveness due to a complicated operation and an inapplicability to an automation, and a further convenient and effective sample preparation method is required.
Non-Patent Document 1: Adam H. Brockman, Brian S. Dodd and Ron Orland, Anal. Chem., 69, 4716-4720 (1997)
Non-Patent Document 2: Maria E. Warren, Adam H. Brockman and Ron Orlando, Anal. Chem., 70, 3757-3761 (1998)
Non-Patent Document 3: Jason C. Rouse and James E. Vath, Analytical Biochemistry 238, 82-92 (1996)
Non-Patent Document 4: Rajendram V. Rajnarayanan and Kuan Wang, J. Mass Spectrum. 39, 79-85 (2004)